Rickettsial diseases, such as epidemic typhus, a louse-vectored disease of humans, are a world-wide scourge for which fundamental understanding of the etiologic agent, better diagnostic tools, and vaccines are needed. The etiologic agent, R. prowazekii, is an obligate intracellular bacterial parasite; rickettsiae only grow directly in the cytoplasm of hosts and vectors. Rickettsiae are marvelous organisms to unravel the biological mysteries of obligate intracellular parasitism and living probes for the investigation of the eukaryotic host cell cytoplasm. R. prowazekii has only 834 genes because many metabolic intermediates need not be synthesized, but are transported from the cytoplasm of the host cell - often by unusual transport systems that have no counterparts in free-living bacteria. The ATP/ADP translocase (Tlc1) of R. prowazekii transports energy via the obligatory exchange of rickettsial ADP for host cell ATP. We now have an excellent two-dimensional topology for this protein and evidence for which transmembrane domains (TMs) form the water-filled channel through which ATP/ADP crosses the membrane. Our current Aim 1 is to elucidate the structure of its 12 TMs, the Tlc1 helix packing. In Aim 2 the substrate-binding site of Tlc1 will be determined through DNA shuffling and formation of chimeric proteins. We will do 'evolution in the test-tube' by shuffling the tlc1 genes of Caedibacter caryophilus and R. prowazekii. Sequence analysis of shuffled fragments within these chimeric proteins will associate conserved sequence motifs with substrate specificity. In Aim 3 we will identify and characterize the transport system of NAD, a principle metabolite in R. prowazekii, and in Aim 4 we will investigate the phospholipase-like proteins, patatin and ExoU, in R. prowazekii. We have demonstrated that interaction of rickettsiae with host cells results in the biochemical products of phospholipase activity. This is involved in one of the most fundamental questions in rickettsial biology: its entry. The hypothesis is that activation and/or translocation is required for enzyme activity and rickettsial entry and we will determine if ExoU and patatin play a role in this process.